Genetic Analyses and Genome-Wide Association Studies on Pathogen Resistance of Bos Taurus and Bos Indicus Cattle Breeds in Cameroon

Home , Faro (department), Vina (department)

G C A T T A C G G C A T genes

Communication Genetic Analyses and Genome-Wide Association Studies on Pathogen Resistance of Bos taurus and Bos indicus Cattle Breeds in Cameroon

Babette Abanda 1,*,† , Markus Schmid 2,†, Archile Paguem 1,3 , Hanna Ifﬂand 2, Siegfried Preuß 2, Alfons Renz 1 and Albert Eisenbarth 1

1 Department of Comparative Zoology, Institute of Evolution and Ecology, University of Tübingen, 72076 Tübingen, Germany; [email protected] (A.P.); [email protected] (A.R.); [email protected] (A.E.) 2 Institute of Animal Science, University of Hohenheim, 70599 Stuttgart, Germany; [email protected] (M.S.); hanna.ifﬂ[email protected] (H.I.); [email protected] (S.P.) 3 Department of Biological Sciences, University of Ngaoundéré, Ngaoundéré, Cameroon * Correspondence: [email protected] † Both authors contributed equally to this manuscript.

Abstract: Autochthonous taurine and later introduced zebu cattle from Cameroon differ considerably in their resistance to endemic pathogens with little to no reports of the underlying genetic make-up. Breed history and habitat variations are reported to contribute significantly to this diversity world- wide, presumably in Cameroon as well, where locations diverge in climate, pasture, and prevalence of infectious agents. In order to investigate the genetic background, the genotypes of 685 individuals Citation: Abanda, B.; Schmid, M.; of different Cameroonian breeds were analysed by using the BovineSNP50v3 BeadChip. The variance Paguem, A.; Iffland, H.; Preuß, S.; components including heritability were estimated and genome-wide association studies (GWAS) Renz, A.; Eisenbarth, A. Genetic were performed. Phenotypes were obtained by parasitological screening and categorised in Tick- Analyses and Genome-Wide Association Studies on Pathogen borne pathogens (TBP), gastrointestinal nematodes (GIN), and onchocercosis (ONC). Estimated Resistance of Bos taurus and Bos heritabilities were low for GIN and TBP (0.079 (se = 0.084) and 0.109 (se = 0.103) respectively) and indicus Cattle Breeds in Cameroon. moderate for ONC (0.216 (se = 0.094)). Further than revealing the quantitative nature of the traits, Genes 2021, 12, 976. https:// GWAS identified putative trait-associated genomic regions on five chromosomes, including the doi.org/10.3390/genes12070976 chromosomes 11 and 18 for GIN, 20 and 24 for TBP, and 12 for ONC. The results imply that breeding for resistant animals in the cattle population from Northern Cameroon might be possible for the Academic Editor: Ottmar Distl studied pathogens; however, further research in this field using larger datasets will be required to improve the resistance towards pathogen infections, propose candidate genes or to infer biological Received: 1 June 2021 pathways, as well as the genetic structures of African multi-breed populations. Accepted: 24 June 2021 Published: 26 June 2021 Keywords: SNP chip; heritability; case-control; parasitic disease; cattle

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- 1. Introduction iations. In cattle breeds, the genetic makeup has been shown to create different phenotypes related to their ability to sustain environmental pressure, including pathogens. Partic- ularly in developing countries, many of the endemic pathogens considerably affect the cattle livestock industry [1], for instance endoparasitic infections caused by a single or Copyright: © 2021 by the authors. mixture of gastrointestinal nematodes (GIN) such as Toxocara, Strongyles, Strongyloides, and Licensee MDPI, Basel, Switzerland. This article is an open access article Trichuris [2,3]. These infections lead to losses in meat and milk production systems. Addi- distributed under the terms and tionally, female infertility and rapid death in younger animals may occur after infection conditions of the Creative Commons without warning signs [4]. The resistance to tick bites via immunogenic reactivity against Attribution (CC BY) license (https:// compounds is in the tick salivary, and thus the likelihood to acquire tick transmissible creativecommons.org/licenses/by/ microorganisms is reported to vary among different breeds of cattle [5,6]. This combination 4.0/). of ticks and transmissible diseases (tick-borne pathogens: TBP) is known to be a substantial

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drawback for the improvement of the livestock sector in sub-Saharan Africa. As an example, in many regions of Africa, the tick species Rhipicephalus (Boophilus) microplus is progres- sively invading new areas with published reports of displacing established populations of closely-related species [7]. Cameroon is a country situated in Central Africa and home to both autochthonous taurine (Bos taurus) and zebu (Bos indicus) cattle breeds, as well as to all agents of the above-mentioned infectious diseases. The respective distribution of those pathogens is thereby dependent on the climatic zone, habitat preference and vector abundance. The whole genomes of individuals of the investigated breeds have been recently published [8]. The latter study revealed hints of considerable breed admixture, possibly due to uncontrolled mating. To assess the possible exploitation of economically valuable phenotypic traits, heritability studies have been used all over the world evaluating which of the related traits may be targets for breeding improvement [9–11]. In Cameroon, heritability studies on cattle resistance to parasites have been done for ticks [12,13], but no clear genetic effect could be demonstrated. In the human host, substantial inter-individual variations of infection burden with the tissue-dwelling ﬁlarial nematode Onchocerca volvulus were not explainable by exposure to parasite transmission, suggesting a substantial genetic predis- position [14]. In the cattle host, exposed but non-Onchocerca ochengi infected individuals have been described as putative immune [15], with no gene association study yet carried out. Although most species of the bovine Onchocerca (ONC) parasites have been reported non- or low-pathogenic [16], their importance mainly being a naturally occurring model for the closely-related human parasite. Traits associated with disease resistance have already been found to be heritable, with identiﬁed regions mapped by genome-wide association studies (GWAS), even if at times with low estimates [9,17]. The present study aims to investigate the genetic background of the resistance against pathogens by using the infection status of individuals with regard to vector-borne (TBP and ONC) and oral-faecal-transmitted (GIN) pathogens in cattle breeds from Cameroon.

2. Materials and Methods 2.1. Sample Collection As large datasets are indispensable for reliable results in genomic studies [18], a pooled multi-breed dataset was examined. A total of 1260 cattle were examined, of which a subset of 719 (57%) was selected for genotyping analyses. Thirty-four samples did not meet the quality control criteria and were excluded for the analysis. The sample set originated from ﬁve districts in North Cameroon located in the provincial regions Adamawa, North and Far North where approximately 83% of the national cattle population is located [19], and consisted of 719 individuals from the Bos indicus breeds Fulani (n = 100) and Gudali (n = 372), as well as the autochthonous Bos taurus breeds Kapsiki (n = 137) and Namchi (n = 110). Additional information is available from [20,21]. The study data collection took place in both dry and rainy seasons of the year 2014. The sampling sites spanned across three different bioclimatic zones: the sub-humid Guinea savannah biotope of the Adamawa highlands, the semi-arid Sudan savannah in the North region, and the arid Sahel region in the region Far North (Figure1). The regions were subdivided into ﬁve sites according to the availability of samples and the willingness of the owner to participate in the study. The number of individuals after/before quality control for each district, respectively, were: Vina (n = 123/125) and Faro et Deo (n = 105/128) in the Adamawa region; Faro (n = 106/110) and Mayo-Rey (n = 215/219) in the North region, and Mayo Tsanaga (n = 136/137) in the Far North region. The quality control ensured that individuals without reliable phenotypic records or more than 10% missing genotypes were discarded. The sample collections have been carried out with the consent of the regional state representatives and traditional authorities from each of the sampling areas. Furthermore, oral consent was given by the cattle owners, herdsmen (who also helped in restraining the animals), and with the participation and approval of the National Institute Genes 2021, 12, x FOR PEER REVIEW 3 of 10

sample collections have been carried out with the consent of the regional state representatives and traditional authorities from each of the sampling areas. Furthermore, oral con- Genes 2021, 12, 976 3 of 10 sent was given by the cattle owners, herdsmen (who also helped in restraining the animals), and with the participation and approval of the National Institute of Agricultural Research for Developmentof Agricultural (IRAD) Researchin Cameroon, for Development which is (IRAD) the country’s in Cameroon, government which is the country’s institution for animal healthgovernment and livestock institution husbandry for animal health improvement. and livestock husbandry improvement.

Figure 1. Sampling. The Vina and Faro et Deo sites are located in the Adamawa region, the Faro and the Mayo-Rey in

the North region, and the Mayo Tsanaga in the Far North region. Ecological zones and climatic conditions of each of the Figuresampling 1. Sampling. zones are shown. The TheVina coloured and Fa zonesro representet Deo sites the sampling are located areas, the in zonesthe Adamawa with stripes the region, national the parks, Faro and andthe the red Mayo dots the-Rey sampling in the sites. North Image region, adapted fromand [the22]. Mayo Tsanaga in the Far North region. Ecological zones and climatic conditions of each of the sampling zones are shown. The coloured zones represent the sampling areas, 2.2.the Pathogenzones with Identification stripes andthe Phenotype national Classification parks, and the red dots the sampling sites. Image adapted from [22].DNA was extracted from blood, as previously reported [20]. Phenotypic information was obtained using molecular detection for TBP, faeces treated by flotation technique for GIN, and small skin biopsies from the inguinal region for ONC. Details of the TBP 2.2. Pathogen Identificationidentification and Phenotype by molecular Classification tool, distribution and biodiversity is given in Abanda et al. [20]. DNA was extractedIsolated from eggs blood of GIN, as werepreviously identified reported by the McMaster [20]. Phenotypic technique [23 ],informationOnchocerca spp. were investigated by intradermal nodule palpation [24] and isolation and identification was obtained using molecularof microfilariae detection of O. ochengi for ,TBP,O. gutturosa faecesand treatedO. armillata by accordingflotation to technique Wahl et al. [16 for] for GIN, and small skin biopsiesONC. All from phenotypes the inguinal were binary region coded as for 1 (infected) ONC. Details and 0 (not of infected). the TBP identification by molecular tool, distribution and biodiversity is given in Abanda et al. [20]. Isolated eggs of GIN were identified by the McMaster technique [23], Onchocerca spp. were investigated by intradermal nodule palpation [24] and isolation and identification of microfilariae of O. ochengi, O. gutturosa and O. armillata according to Wahl et al. [16] for ONC. All phenotypes were binary coded as 1 (infected) and 0 (not infected).

2.3. Genotyping Genotyping was conducted using the Illumina BovineSNP50v3 BeadChip (Illumina, San Diego, CA, USA). After quality control, all annotated autosomal single nucleotide

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2.3. Genotyping Genotyping was conducted using the Illumina BovineSNP50v3 BeadChip (Illumina, San Diego, CA, USA). After quality control, all annotated autosomal single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) ≥ 0.05, no signiﬁcant devi- ation from Hardy-Weinberg-equilibrium (p < 0.001), and segregating in all of the breeds were favoured for downstream analyses. The ﬁnal dataset contained the phenotypes of 608 to 685 animals depending on the trait and their genotype status of 35,195 SNPs (Supplementary Table S1).

2.4. Statistical Analysis The statistical analysis consisted of two major sections, the estimation of variance components, and the estimation of marker effects in order to infer marker-trait-association. Both were conducted using the software GCTA (version 1.91.7beta) [25]. Initially, all available ﬁxed effects (sex, age, season_year, breed_site) were tested for signiﬁcance (p < 0.05) to determine the effects to be included in the evaluation model. Since not all breeds were present at all sites, and samples were not taken in all seasons in each year, combined effects (breed_site and season_year) were considered. The heritabilities were calculated based on the estimated variance components using standard notations. For the variance component analyses, the following model was applied:

y = Xb + g + e (1)

where vector y contains the phenotypes of the individuals, b denotes the ﬁxed effect breed_site (additionally age for the trait ONC), and X is the corresponding design matrix. 2 g is the random genetic animal effect, with g ∼ N 0, Gσg and G being the genomic 2 relationship matrix. The vector e includes the residuals, with e ∼ N 0, Iσe , where I is the identity matrix. As the traits were binary coded, heritabilities were calculated on the observed, as well as on the unobserved liability scale (λ), and given assumed population prevalences of 0.8, 0.6 and 0.5 for the traits GIN, ONC, and TBP, respectively. Thereby, λ is obtained by linear transformation of the observed binary coded phenotype to an unobserved continuous scale. In order to estimate the level of association between the traits and the signiﬁcant SNPs in GWAS, model 1 was extended towards:

y = Xb + Wu + g + e (2)

Thereby, u denotes the fixed effect of the SNP to be tested and W the design matrix containing the number of 1-alleles. A leave-one-chromosome-out (loco) approach was applied to avoid a loss in mapping power by double-fitting the tested SNP. Due to the limitation of the sample size and the associated produced dataset, a Bonferroni corrected genome-wide significance level was too stringent to be applied, hence SNPs with p-values −5 smaller than the threshold of pnominal = 5 × 10 were assumed to show nominal significant 0.05 trait association. The genome-wide significance level was defined as pgenome−wide = n , where n denotes the number of SNPs.

3. Results Parasitological examinations representing phenotypic data are reported in Table1 including the cattle breed, the pathogen considered, and the respective distribution according to the site of sampling for TBP, ONC, and GIN. The fixed effect breed_site had a significant impact on all traits whereas the age-effect was solely significant for ONC. Genes 2021, 12, 976 5 of 10

Table 1. Characteristics of phenotyped and genotyped dataset used in the analysis, including the sampling site and parasite burden.

GIN 1 ONC 2 TBP 3 Site Cattle Breed n Species + NA - + NA - + NA - Mayo Tsanaga Kapsiki 136 B. taurus 118 8 10 110 0 26 134 0 2 Faro Namchi 106 B. taurus 80 0 26 52 1 53 95 0 11 Mayo Rey Fulani 26 B. indicus 25 0 1 24 0 2 21 0 5 Gudali 189 B. indicus 188 0 1 149 2 38 169 0 20 Vina Gudali 123 B. indicus 117 0 6 75 7 41 116 0 7 Faro et Deo Fulani 68 B. indicus 47 2 19 1 66 1 67 0 1 Gudali 37 B. indicus 26 0 11 11 1 25 36 0 1 TOTAL 242 B. taurus 198 8 36 162 1 79 229 0 13 443 B. indicus 403 2 38 260 76 107 409 0 34 685 601 10 74 422 77 186 638 0 47 1 gastrointestinal nematodes, identified by McMaster faeces flotation: Toxocara spp., Strongyle spp., Strongyloides spp., Trichuris spp. 2 onchocercosis, identified by skin biopsies and/or palpation *: Onchocerca ochengi *, O. gutturosa, O. dukei, O. armillata. 3 tick-borne pathogens, identified by PCR: Anaplasma spp., Borrelia spp., Rickettsia spp., Theileria spp. +: tested positive; NA: test result not available; -: tested negative; * indicates palpation. All species marked with asterisk were detected by palpation. The rest only by skin biopsies.

The estimated variance components, as well as heritabilities based on the observed and the liability scale are given in Table2. Phenotypic variances with low standard errors were estimated ranging from 0.063 to 0.181. A high or low observed pathogen prevalence in the population came along with considerably smaller estimates. The greatest phenotypic variance was by far observed for ONC, for which the prevalence was rather close to an intermediate value. Analogous results could be observed for the estimated additive genetic variance resulting in low heritabilities on the observed scale for all traits but ONC 2 (hobs. = 0.216). The standard errors were generally large for the estimates of the additive genetic variance and the heritabilities. The heritability estimates were substantially higher when the estimation was based on the liability scale and resulted in moderate to high heritabilities for all investigated traits. However, the standard errors were large and even exceeded the estimates in the case of GIN.

Table 2. Population speciﬁc parameters of the investigated traits. The estimated phenotypic (VP) 2 and additive genetic (VA) variance, the heritability estimated on the observed (hobs.) and liability 2 scale (hliab.) as well as their standard errors (in parentheses) are shown. The number of evaluated individuals (n) and the observed prevalence in the investigated population are given.

1 2 2 Trait n Prevalence VP (SE) VA (SE) hobs. (SE) hliab. (SE) 0.087 0.006 0.079 0.265 GIN 675 0.890 (0.005) (0.007) (0.084) (0.281) 0.181 0.039 0.216 0.393 ONC 608 0.694 (0.011) (0.017) (0.094) (0.170) 0.063 0.007 0.109 0.666 TBP 685 0.931 (0.003) (0.006) (0.103) (0.631) 1 gastrointestinal nematodes (GIN), onchocercosis (ONC), tick-borne pathogens (TBP).

Figure2 displays the GWAS results for each of the studied traits. For all analysed traits, the majority of SNP effects were small; however, several regions harbouring SNPs with obviously larger effects were distributed across almost all chromosomes. Two SNPs on chromosome 11 and 18 exceeded the nominal significance threshold indicating putative trait-associated genomic regions for GIN (Figure2A), and one on chromosome 12 for ONC (Figure2B). For TBP (Figure2C), one significant SNP was found on chromosome 20 and another one on chromosome 24. None of the reported outlying SNPs reported from the traits reached the genome-wide level of significance. GenesGenes2021 2021, 12, ,12 976, x FOR PEER REVIEW 66 of of 10 10

A B C

Figure 2. Manhattan plots for the investigated traits. The −log10-p-values of the SNP effects and their chromosomal Figure 2. Manhattan plots for the investigated traits. The −log10-p-values of the SNP effects and their chromosomal posi- positionstions are are shown shown for for the the traits traits gastrointestinal gastrointestinal nematodes nematodes (A) (,A onchocercosis), onchocercosis (B) (andB) and tick tick-borne-borne pathogens pathogens (C). ( CThe). The hori- − horizontal bottom black line corresponds to a nominal signiﬁcance level of p = 5 × 10−5 5, the top red line to the zontal bottom black line corresponds to a nominal significance level of 푝푛표푚푖푛푎푙nominal= 5 × 10 , the top red line to the ge- genome-widenome-wide significance signiﬁcance level.

4.4. Discussion Discussion AsAs in in many many parts parts of of the the world, world, the the distribution distribution and and phenotypic phenotypic adaptation adaptation of of cattle cattle inin Cameroon Cameroon has has been been strongly strongly influenced influenced by by herd herd migration migration history, history, climate, climate, wildlife wildlife abundance,abundance, vector vector and and pathogen pathogen occurrence, occurrence, and and food food and and water water accessibility accessibility [ 26 [26–29–29].]. OurOur results results showed showed that that the the fixed fixed effect effect breed_site breed_site had had a significanta significant association association with with the the acquisitionacquisition of of all all investigated investigated pathogens.pathogens. This This was was expected expected because because the the studied studied sites’ sites’ cli- climatemate differed differed widely widely from from humid humid to to sub sub-humid-humid and and arid arid conditions conditions [29,30]. [29,30 ].Further, Further, ge- geneticnetic studies studies observed observed differences between BosBos taurustaurus andandBos Bos indicus indicusbreeds breeds and and have have shownshown that that genetic genetic variation variation plays plays a a significant significant role role in in terms terms of of an an animal’s animal’s resistance resistance towardstowards parasites parasites as reviewedas reviewed by Mapholiby Mapholi et al. et [31 al.,32 [31,32].]. Given Given the present the present dataset, dataset, the effect the ofeffect the breed of the and breed the siteand couldthe site not could be distinguished not be distinguished due to the due heterogeneous to the heterogeneous distribution dis- oftribution all breeds of not all presentbreeds not at everypresent site. at Consequently,every site. Consequently, breeds were breeds not separately were not analysedseparately as large datasets are needed to obtain reliable results in genomic studies, and pooling analysed as large datasets are needed to obtain reliable results in genomic studies, and data being a suitable approach to increase the power in such studies and to strengthen the pooling data being a suitable approach to increase the power in such studies and to results [18]. The observed significance of the age effect in the acquisition of ONC can be strengthen the results [18]. The observed significance of the age effect in the acquisition of explained by the cumulative character of the long-living parasite population over time. To ONC can be explained by the cumulative character of the long-living parasite population prove that statement in the present study, we grouped all individuals according to their over time. To prove that statement in the present study, we grouped all individuals ac- age (<3 years, >3 years) and calculated the prevalence of infection within these groups. cording to their age (<3 years, >3 years) and calculated the prevalence of infection within Thereby, 73% of the individuals that were older than 3 years were infected, whereas younger these groups. Thereby, 73% of the individuals that were older than 3 years were infected, individuals were less affected (46%). This evidence has been previously demonstrated in whereas younger individuals were less affected (46%). This evidence has been previously Gudali cattle from the Adamawa region in Cameroon [33]. Indeed, susceptible individuals demonstrated in Gudali cattle from the Adamawa region in Cameroon [33]. Indeed, sus- grouped into early and late susceptible had increasing nodules and microfilarial loads with ceptible individuals grouped into early and late susceptible had increasing nodules and significant differences in the prepatency period [24,33] variable from male to female, but withmicrofilarial no visible loads gender with effect significant in our dataset. differences in the prepatency period [24,33] variable from male to female, but with no visible gender effect in our dataset.

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The variance component estimation uncovered the susceptibility of individuals towards infections with the studied pathogens to be to some extent controlled by genetics. As expected for binary coded traits, a high or low observed pathogen prevalence in the population came along with considerably smaller variance component estimates [34]. Compared with the other traits, obviously larger variance component estimates could be presented for ONC, for which the pathogen prevalence was rather close to an intermediate observed prevalence. In agreement with [35], all heritabilities were substantially higher when the estimation was based on the liability scale since these also capture parts of non-additive genetic variance, particularly for traits where the prevalence is close to zero or unity [34]. The standard errors were generally large for the estimates of the additive genetic variance and the heritabilities, mainly since the number of individuals was limited and the multi- breed data structure was complex. The results imply that breeding might be possible to improve pathogen resistance in Cameroonian cattle for all three traits investigated here. However, as the variance components are population-specific parameters and are additionally influenced by the prevalence within a population [36], further studies using large representative datasets of the breeds are required to accurately determine the extent of the genetic variation or the response to selection within certain breeds. The visualised GWAS results (Figure2) are characterised by numerous small and a few larger SNP effects across all chromosomes implying a quantitative trait nature for each of the traits. Several putatively trait-associated regions were identified for all of the traits. The region located on chromosome 24 having an impact on TBP has not been reported as an association signal elsewhere [37]. Generally, for all traits investigated, a relatively small number of neighbouring SNPs seems to be in strong linkage disequilibrium (LD) with the significant SNPs, which in some cases led to single significant SNPs instead of clear peaks, i.e., clear association signals (see Figure2). This might be attributed to the multi-breed dataset, for which a large effective population size can be assumed and hence LD decays fast [38,39]. Additionally, due to data filtering, the number of SNPs can be decreased in chromosomal regions where an increased number of SNPs do not segregate in all of the breeds. As a Bonferroni corrected genome-wide significance level is too stringent, especially when the tests are not independent, like in the present study, an additional nominal significance level was applied to detect regions with weak associations. Due to the limited statistical power resulting in a lack of genome-wide significant SNPs, post-GWAS analyses were not conducted. In order to actually map and discuss quantitative trait loci (QTL), larger sample sizes are required. Hence no further discussion of the association signals was intended here. A plethora of constantly updated reports [17,40,41] of SNPs associated with TBP and GIN, as well as the heritability estimates between low to medium values in the present study (see Table2) confirm the importance of genomics for livestock improvement. Even though this study has discovered findings of such potential, they should be interpreted with caution due to the large standard errors of the estimates. In future investigations, large-scale studies or meta-analyses might give a better insight into the architecture of the traits in future GWAS. Special attention should be payed to LD structures in multi- breed populations as investigated here because LD consistency is compromised across populations [42]. In summary, the results imply that breeding for resistant animals in the cattle population from Northern Cameroon might be possible for the studied pathogens. Nevertheless, the findings suggest that further research in this field using larger datasets will be worthwhile to improve the resistance towards pathogen infections and to infer the genetic structures of African multi-breed populations. The latter is of great importance because uncontrolled admixture is predominant in the breeds investigated [8]. In fact, individual herd performances of local adaptation and resistance to pathogens can greatly vary based on the management system and the level of introgressed genes. It is therefore recommended to conduct interdisciplinary studies where genetic parameters are supplemented with environmental factors, information of the husbandry system, and Genes 2021, 12, 976 8 of 10

molecular-diagnostic analyses for local pathogens diversity and exposure, together with productivity determinants.

5. Conclusions The present study is the ﬁrst in-depth investigation of the genetic makeup towards pathogen resistance of the cattle population in Cameroon. For all the studied groups of endemic pathogens genetic determinants were found, but due to the combination of high population variability and the limited sample size, a clear association with the involved genes was not possible. Future genetic association studies should consider these circumstances already in their planning phase.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/genes12070976/s1, Table S1: SNP profile of the analysed cattle generated by the Illumina Bovine SNP50v3 chip. Author Contributions: Conceptualization, A.E., B.A., A.P. and A.R.; methodology, A.E., B.A., A.P., S.P., M.S. and H.I.; software, B.A., M.S., H.I. and S.P.; validation, A.E., B.A., M.S., H.I. and S.P.; formal analysis, H.I.; investigation, B.A. and A.P.; resources, A.E., B.A. and A.P.; data curation, A.E., B.A., M.S. and H.I.; writing—original draft preparation, B.A. and M.S.; writing—review and editing, B.A., H.I.,M.S. and A.E.; visualization, A.E., M.S. and H.I.; supervision, A.E. and A.R.; project administration, A.E.; funding acquisition, A.E., B.A., A.P. and A.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the joint RiSC program of the State Ministry of Science, Research and Arts Baden-Württemberg and the University of Tübingen (PSP-no. 4041002616). We are thankful for the use of de.NBI cloud and the support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the University of Tübingen and the Federal Ministry of Education and Research (BMBF) through grant no. 031 A535A. The sample collections and parasitological examinations were supported by the Otto Bayer Foundation (grant no. F-2013BS522) and the German Research Foundation (DFG, grant no. Re 1536/2-1). The research stays of B.A. and A.P. in Germany were supported by the Baden-Württemberg Foundation and additionally for B.A. the German Academic Exchange Service (DAAD, grant no. BA_A/12/97080). We acknowledge support on publication charges (APC) by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of University of Tübingen. Institutional Review Board Statement: The study was conducted according to the guidelines of the National Institute of Agricultural Research for Development (IRAD) in Cameroon, which is the country’s government institution for animal health and livestock husbandry improvement. The sample where collected as regular screening for veterinary intervention and parasitological assessment. Veterinarians and experienced personal where involved. The presents research is the 4th of a series of four publications (3 already published) using the same individuals to reports the epidemiology of the screened pathogens in the targeted population where the Institutional Review Board Statement is equally stated. No ethical code number was granted to the inspection, as regularly undertaken for screening purposes. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. For further enquiries contact the corresponding author here [email protected]. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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